Enzymes which metabolize nucleic acids in a manner specified by primary sequence, backbone structure, or a base character (often damaged or modified base) are of utility in biotechnology applications. Several families of such enzymes are used routinely in nucleic acid-based techniques and include restriction endonucleases, polymerases, ligases and exonucleases. Additionally, a variety of single-subunit (non-restriction) endonucleases which rely not on specific sequence strings but on recognising unusual, damaged or missing bases have been described over the years. These enzymes can be loosely divided into 2 groups—the AP endonucleases, of which E. coli endonuclease IV (Nfo) and E. coli exonuclease III are examples, and the DNA glycosylase/lyase family of which E. coli fpg, MUG and Nth are examples.
The AP endonucleases are characterised by the ability to recognise and cleave the sugar-phosphate backbone at abasic sites (other enzymatic activities may also be present) when found in the context of duplex DNA. Recognition and incision at abasic sites occurs in a biochemical manner that is distinct to the glycosylase/lyase family and not by beta-elimination or beta/delta-elimination. Consequently they attack not only true abasic sites but other substrates including tetrayhydrofuran moieties which lack an oxygen atom on the 1′ carbon of the sugar ring (Takeshita et al., 1987, J Biol Chem. 262(21):10171)(see FIG. 1 for chemical structures).
In contrast, glycosylase/lyase enzymes including the fpg protein (8-oxoguanine DNA glycosylase, fpg in E. coli and OGG1 in mammals) or Nth proteins (endonuclease III in E. coli, Nth1 in humans, etc.) function in a 2-stage catalytic manner in which damaged bases are first recognized and excised via formation of a Schiff base between the protein and the DNA, and secondly the abasic site thus generated is processed by beta-elimination or beta-delta elimination in a manner distinct to the AP endonucleases. In this case tetrahydrofuran (THF) residues are not a substrate for lyase activity as no C1′ oxygen atom is present in this abasic mimic and such sugars lacking oxygen at the 1′ position are resistant to attack (Takeshita et al., 1987)(FIG. 1).
The use of AP endonucleases and glycosylase/lyases in molecular biology techniques has been described. One application is the use of these enzymes to process substrates generated during in vitro DNA amplification reactions, or similar kinds of applications, and in particular when a synthetic ‘probe’ oligonucleotide has been provided containing modified sugars or bases which can become a substrate for the enzymes if the synthetic oligonucleotide hybridizes specifically to molecules in the sample. An example of such an application is given in U.S. Pat. No. 7,435,561 B2 and Piepenburg et al., Plos Biology, 2006 4(7):e204 in which tetrahydrofuran-modified oligonucleotides are used as substrates for the E. coli Nfo (endonuclease IV) protein as a method to measure DNA amplification (Nfo is one of the two AP endonucleases of E. coli).
Application of glycosylase/lyases to similar strategies can also be envisioned. The ability of fpg protein to similarly process modified bases such as 8-oxoguanine within a DNA amplification reaction for the purposes of reaction-monitoring has been described (U.S. Pat. No. 7,435,561 B2). Furthermore the fact that glycosylase/lyase enzymes such as fpg and Nth do not leave 3′ extendable ends but rather blocked 3′ ends (due to the differences in catalytic mode) may have particular utility in circumstances in which one wishes to ensure that the processed probe cannot be a ready substrate for polymerases or other activities dependent on a 3′ hydroxyl moiety.
Despite the potential of these enzymes, they possess certain features that make them unattractive for use in some applications. Notably, unlike the THF residue, true abasic sites required for the backbone-incising activity of DNA lyases are not stable under physiological conditions and are quickly hydrolyzed in aqueous solutions making them impractical for use in most molecular procedures. Instead specific damaged bases can be incorporated and used as the primary substrates for the glycosylase activity to generate the abasic site transiently before backbone hydrolysis by the lyase activity. Unfortunately however, typical damaged base analogs such as 8-oxoguanine (fpg) or thymidine glycol (Nth) tend to be rather expensive to synthesize and also impart sequence requirements on the probe as ideally they must be paired opposite specific bases on the opposing strand. In principle it would be far more convenient to have a stable substrate analogous to the generic THF residue that can be employed for AP endonucleases but retaining reactivity with the lyase activity of glycosylase/lyase enzymes.
Here we show that the fpg protein, as well as the AP endonuclease IV of E. coli (Nfo), efficiently cleaves DNA backbones containing a variety of substrates that lack a base but contain a 1′-oxygen atom covalently attached to a carbon-based linker [C]n. The linker can itself be used to attach other moieties such as biotin, fluorophores and other coupled groups, particularly useful if an amine-ended linker can be used to couple a variety of agents. Surprisingly, nucleotides having this arrangement and referred to generally as dR-O—[C]n appear to be good substrates of the fpg protein in a number of contexts, and are also substrates for the endonuclease IV protein, but appear relatively poor substrates for E. coli exonuclease III. We anticipate the use of oligonucleotides containing such dR-O—[C]n groups as substrates in a number of circumstances, in particular within in vitro reactions such as part of detection strategies for nucleic acid detection methods. The length of the linker used in this study is 6 carbon atoms, as available on certain commercially available nucleotides, however it is anticipated that a variety of carbon chain lengths might be employed and that it is the carbon-oxygen-carbon structure with little subsequent steric bulking that affords these structures sufficient plasticity to the enzymes.